WO2018040535A1 - Fgf1突变体和给药方法 - Google Patents

Fgf1突变体和给药方法 Download PDF

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WO2018040535A1
WO2018040535A1 PCT/CN2017/076266 CN2017076266W WO2018040535A1 WO 2018040535 A1 WO2018040535 A1 WO 2018040535A1 CN 2017076266 W CN2017076266 W CN 2017076266W WO 2018040535 A1 WO2018040535 A1 WO 2018040535A1
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fgf1
mutant
medicament
mice
nasal
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PCT/CN2017/076266
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French (fr)
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黄志锋
李校堃
周洁
宋林涛
赵龙伟
陈紫璐
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黄志锋
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of protein technology, and in particular, the present invention relates to a FGF1 mutant which reduces FGF1 mitogenic and proliferative side effects, but has activity such as hypoglycemic activity.
  • FGF Fibroblast Growth Factor
  • FGFR FGF receptor
  • FGF1 can produce important hypoglycemic and insulin-suppressing functions in type 2 diabetic mouse models such as db/db, ob/ob, etc., and even low doses do not cause hypoglycemia (see Nature Review Drug Discovery, 2016; Nature Medicine, 2016; Proc Natl Acad Sci U S A, 2016; Diabetes, 2016; Nature, 2015, etc.).
  • FGF1 acts as a paracrine protein, which forms a tight ternary complex by interacting with heparin and FGFR to initiate a series of downstream complex signaling pathways, including the effects of cell division and proliferation, thus frequent injections. FGF1 will undoubtedly have hidden dangers in the body to induce proliferation and even tumors.
  • FGF1 ⁇ HS heparin binding site of the site mutant
  • FGF1 ⁇ HS heparin binding site of the site mutant
  • the technical problem to be solved by the present invention is to provide a novel FGF1 mutant which substantially eliminates the ability of wild-type FGF1 to promote cell division and proliferation, but still retains the function of lowering blood glucose and the like of FGF1, so that it can be administered safely for a long period of time. Further, the present invention also provides a coding gene, an expression vector, and a host cell of the mutant, and a medical use and the like.
  • the invention provides a FGF1 mutant having an amino acid sequence (only) having a Lys127, Lys128 and/or Lys133 mutation on the basis of SEQ ID NO:1.
  • the mutant of the first aspect of the invention reduces (preferably eliminates) the ability of wild-type FGF1 (whose amino acid sequence is as shown in SEQ ID NO: 1) to promote cell division and proliferation, but still retains functions such as blood glucose lowering.
  • the representation of the mutation is in a manner well known to those skilled in the art.
  • Lys127 indicates mutation of Lys at position 127
  • Lys128Gln indicates mutation of Lys at position 128, which is mutated to Gln.
  • Mutations can be additions, deletions, and/or substitutions. It is known to those skilled in the art that a polypeptide or protein substituted, added and/or deleted with an amino acid residue can be prepared by changing the coding sequence of a known polypeptide and introducing it into an expression vector, which are widely described in the molecular cloning experiment. Guides and the like are well known in the art.
  • the mutation is a substitution.
  • the mutant of the first aspect of the invention (only) has a Lys127Asp, Lys128Gln and/or Lys133Val mutation.
  • the mutant (only) has Lys127Asp, Lys128Gln and Lys133Val mutations.
  • the invention provides a polynucleotide encoding a mutant of the first aspect of the invention.
  • the polynucleotide may be in the form of DNA or in the form of RNA, preferably in the form of DNA.
  • the DNA form includes natural cDNA and synthetic cDNA, and the DNA may be a coding strand or a template strand.
  • a nucleic acid molecule polynucleotide encoding a mutant of the present invention or a fragment thereof can be easily obtained by a person skilled in the art by a conventional technique such as a PCR method, a recombinant method or a synthetic method. Once obtained, these sequences can be cloned into a vector, transformed or transfected into corresponding cells, and then propagated by conventional host cells to isolate a large number of nucleic acid molecules therefrom.
  • the invention provides a vector comprising the polynucleotide of the second aspect of the invention.
  • vectors include expression vectors and cloning vectors, which refer to bacterial plasmids, cosmids, and phages commonly used in the art. Granules, yeast plasmids, plant cell viruses, animal viruses, and various other viral vectors.
  • Vectors suitable for use in the present invention include, but are not limited to, a vector for expression in bacteria (prokaryotic expression vector), a vector for expression in yeast (e.g., Pichia vector, Hansenula vector, etc.), expressed in insect cells.
  • Baculovirus vector vector for expression in mammalian cells (vaccinia virus vector, retroviral vector, adenovirus vector, adeno-associated virus vector, etc.), plant viral vector for expression in plants, and mammalian mammary gland Various vectors for expression in the medium.
  • any plasmid and vector can be used as long as it is stable in host cells.
  • the expression vector comprises a selectable marker gene, such as a bacterial ampicillin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a streptomycin resistance gene, a chloramphenicol resistance gene; a yeast neomycin Resistance gene, Zeocin resistance gene, yeast selection marker of defects, such as His, Leu, Trp, etc.; neomycin resistance gene, Zeocin resistance gene, dihydrofolate reductase gene and fluorescent protein marker of eukaryotic cells Genes, etc.
  • a commercially available pET plasmid is used.
  • the invention provides a cell, such as a host cell, comprising a polynucleotide of the second aspect of the invention.
  • the cells of the present invention may contain the vector of the third aspect of the invention, or may be transformed or transfected with the polynucleotide of the second aspect of the invention.
  • the host cell may be a prokaryotic cell or a eukaryotic cell, such as a bacterial cell, a yeast cell, a plant cell, an insect cell, a mammalian cell, or the like.
  • the host cell after transformation or transfection of the gene sequence comprising the protein encoded by the invention, constitutes an engineered cell or cell line and can be used to produce the desired fusion protein.
  • the host cell of the invention is Escherichia coli BL21 (DE3).
  • the present invention provides a pharmaceutical composition comprising the mutant of the first aspect of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention is capable of treating diabetes, lowering blood sugar, treating or preventing obesity, anti-inflammatory or for reducing inflammatory cytokine levels, and it reduces (or even eliminates) the side effects of FGF1 promoting cell proliferation.
  • a pharmaceutically acceptable carrier refers to a non-toxic filler, stabilizer, diluent, adjuvant or other formulation adjuvant.
  • the pharmaceutical compositions can be formulated into a variety of dosage forms, including dry powders (eg, lyophilized powder, depending on the purpose of the treatment, the route of administration, according to techniques well known in the art) Agents, tablets, capsules, drops, emulsions, injections and sprays, and the like.
  • the pharmaceutical composition may be administered brain, and thus it may be a preparation for brain administration such as a brain injection.
  • the administration route includes injection and nasal administration, and therefore the pharmaceutical composition of the fifth aspect of the invention is an injection or a nasal preparation.
  • the nasal administration preparation comprises a liposome-encapsulated mutant of the first aspect of the invention, which may be a nasal spray or a nasal drop.
  • the nasal spray of the present invention may be a liposome-coated lyophilized powder of the mutant of the first aspect of the invention (or wild-type FGF1).
  • the liposome-encapsulated mutant of the first aspect of the invention (or wild-type FGF1) has a particle size between 100 nm and 1000 nm in the lyophilized powder.
  • the lyophilized powder can be prepared by the following method:
  • the mutant of the first aspect of the invention (or wild-type FGF1) is dissolved in an aqueous solution of a polymeric hydrophilic gel material, and then poloxamer is added. After mixing uniformly, freeze-drying to form a lyophilized powder; dissolving the lipid material and polyoxyethylene hydrogenated castor oil in tert-butanol; and, adding the above lyophilized powder to the t-butanol solution, dispersing uniformly and then placing The liquid nitrogen freezes to form a solid, which is then freeze-dried to obtain a dry powder.
  • the weight ratio of the mutant (or wild type FGF1), the polymer hydrophilic gel material, the poloxamer, the lipid material and the polyoxyethylene hydrogenated castor oil of the first aspect of the invention is 0.5 to 50: 0.1 ⁇ 10:10 to 1000:0.5 to 50:5 to 500, preferably 1 to 5:0.5 to 2:50 to 200:1 to 5:20 to 100, and most preferably 5:1:100:5:50.
  • the nasal drops of the present invention can be prepared by resuspending the above lyophilized powder in a pharmaceutically acceptable nasal drop solvent.
  • the pharmaceutical composition of the fifth aspect of the invention is preferably in unit dosage form, such as a single, a drop, a needle or a capsule comprising a unit dose of the mutant of the first aspect of the invention.
  • a unit dose refers to the amount of drug required for a single administration of a patient.
  • the pharmaceutical composition of the fifth aspect of the present invention is a nasal preparation having a unit dose of 0.1 to 5 mg, preferably 0.3 to 1 mg, such as 0.5 mg.
  • the hypoglycemic effect of the pharmaceutical composition lasts for at least one week, at least two weeks, or at least one month.
  • the pharmaceutical composition of the fifth aspect of the present invention may also be a preparation for brain administration, and the unit dose thereof is 1 to 500 ⁇ g, preferably 3 to 100 ⁇ g, for example, 10 ⁇ g.
  • the invention provides the use of a mutant of the first aspect of the invention for the manufacture of a medicament for reducing the mitogenic and proliferative side effects of FGF1.
  • Wild-type FGF1 has a role in promoting cell division and proliferation, which easily leads to hyperplasia and even canceration.
  • the mutants of the present invention and their drugs can significantly reduce (or even substantially eliminate) such Side effects, so that it can be administered safely for a long time.
  • the medicament is the pharmaceutical composition of the fifth aspect of the invention.
  • the mutant of the present invention and its drug substantially retain the hypoglycemic function of FGF1. Therefore, preferably in the application of the sixth aspect of the invention, the medicament is for lowering blood sugar. Also preferably in the application of the sixth aspect of the invention, the medicament is for the treatment of diabetes, especially type 2 diabetes.
  • the medicament is for lowering blood fat (e.g., triglyceride). Also preferably in the application of the sixth aspect of the invention, the medicament is for the treatment or prevention of obesity.
  • blood fat e.g., triglyceride
  • the mutants of the present invention and their medicaments also have an anti-inflammatory effect and can be used to lower the levels of some inflammatory cytokines. Therefore, it is preferred that in the application of the sixth aspect of the invention, the medicament is used for anti-inflammatory. Also preferably in the use of the sixth aspect of the invention, the medicament is for reducing the level of inflammatory cytokines (e.g., IL-6 and/or TNF-a).
  • inflammatory cytokines e.g., IL-6 and/or TNF-a
  • the present invention provides a method of continuously lowering blood glucose in a diabetic patient comprising administering to the patient a unit dose of the mutant of the first aspect of the invention once orally or brain.
  • a nasal dose of wild-type FGF1 to a patient with diabetes can also continue to lower blood glucose in this patient.
  • nasal administration can be carried out by nasal spray or nasal drops, more preferably, the unit dose for nasal administration is 0.1 to 5 mg, preferably 0.3 to 1 mg, such as 0.5 mg. .
  • the unit dose for brain administration is 1 to 500 ⁇ g, preferably 3 to 100 ⁇ g, for example, 10 ⁇ g.
  • the mutant of the first aspect of the invention may be continuously hypoglycemic for a long period of time by nasal or cerebral administration, preferably for at least one week, at least two weeks or at least one month.
  • the diabetes is type 2 diabetes.
  • the beneficial effects of the present invention are that the mutant of the present invention substantially loses the function of promoting cell division and proliferation, and at the same time retains the pharmacological action such as hypoglycemic, so it is more safe than the already established FGF1; the basic retention of FGF1
  • the overall structure (without missing the entire HS binding region), so the existing FGF1 genetic engineering production system (including vector, cell and expression conditions) can be utilized as much as possible to facilitate large-scale production; especially through nasal administration Continuous blood sugar lowering, so its nasal administration preparations have become the prospect of becoming a marketed drug.
  • Figure 1 shows the results of various in vitro stimulation of FGF1 WT and FGF1 ⁇ HS , wherein (A) is FGF1 WT , FGF1 ⁇ HS and wild-type FGF21 (FGF21 WT ) in 3T3-L1 adipocytes to FGFR, FGFR bottom Immunoblotting map of activation of substance 2 (FRS2) and MAPK pathway (ERK1/2); (B) FGF1 WT , FGF1 ⁇ HS and wild-type FGF19 (FGF19 WT ) in rat hepatoma cell line H4IIE versus FGFR , immunostaining map of activation of FGFR substrate 2 (FRS2) and MAPK pathway (ERK1/2); (C) cellular glucose uptake by 3GF3-L1 adipocytes stimulated by FGF1 WT , FGF1 ⁇ HS and FGF21 WT (D) FGF1 WT , FGF1 ⁇ HS and FGF19 WT stimulated the cellular glucose
  • Figure 2 shows the mitogenic activity of FGF1 WT and FGF1 ⁇ HS on liver tissue in vivo, wherein (A) is a microscopic examination of rat liver tissue stained with proliferating cell nuclear antigen (PCNA) and Ki67; (B) is FGF1 Immunoblot analysis of liver tissue protein expression in WT and FGF1 ⁇ HS long-term administered mice, *p ⁇ 0.05 vs control (administered with PBS), #p ⁇ 0.05 vs FGF1 WT .
  • PCNA proliferating cell nuclear antigen
  • Figure 3 shows the effects of FGF1 WT and FGF1 ⁇ HS on blood glucose and insulin sensitivity in db/db mice, wherein (A) is a single injection of FGF1 WT and FGF1 ⁇ HS for 24-hour blood glucose in db/db mice. Level effect, *p ⁇ 0.05 vs db/m; #p ⁇ 0.05 vs db/db.
  • FIG. 1 shows dose-dependent effects of FGF1 WT and FGF1 ⁇ HS on blood glucose reduction in db/db mice;
  • the blood glucose level of GTT was 6 hours after a single injection of FGF1 WT and FGF1 ⁇ HS
  • (E) is the blood glucose level of ITT 6 hours after a single injection of FGF1 WT and FGF1 ⁇ HS
  • the AUC of the ITT performed *p ⁇ 0.05 vs db/m; #p ⁇ 0.05 vs db/db
  • (G) is the blood glucose level of db/db mice administered with FGF1 WT and FGF1 ⁇ HS over 28 days, *p
  • Figure 4 shows the long-term effects of FGF1 WT and FGF1 ⁇ HS on liver lipid and glucose metabolism in db/db mice, wherein (A) is a microscopic examination of rat liver tissue stained with H&E, Oil Red O and PAS; (B) Triglyceride content in liver tissues of db/db mice administered with FGF1 WT and FGF1 ⁇ HS over 28 days, (C) db administered with FGF1 WT and FGF1 ⁇ HS over 28 days Glycogen content in liver tissue of /db mice, *p ⁇ 0.05 vs db/m; #p ⁇ 0.05 vs db/db; (DG) shows immunoblot analysis of liver protein expression, (HJ) shows Real-time quantitative PCR analysis of liver mRNA expression, *p ⁇ 0.05 vs db/m; #p ⁇ 0.05 vs db/db.
  • Figure 5 shows the long-term effects of FGF1 WT and FGF1 ⁇ HS on adipose tissue remodeling and lipid metabolism in db/db mice, wherein (A) is a microscopic examination of mouse white adipose tissue (WAT) stained with H&E; (B) is a microscopic quantitative analysis of mouse brown adipose tissue (BAT) stained with H&E; (CG) shows real-time quantitative PCR analysis of WAT mRNA expression, *p ⁇ 0.05 vs db/m; #p ⁇ 0.05 vs db /db.
  • Figure 6 shows the long-term effects of FGF1 WT and FGF1 ⁇ HS on systemic and hepatic inflammation in db/db mice, wherein (A) shows plasma of db/db mice administered with FGF1 WT and FGF1 ⁇ HS over 28 days.
  • the concentration of IL-6 in the medium shows the concentration of TNF- ⁇ in the plasma of db/db mice administered with FGF1 WT and FGF1 ⁇ HS for 28 days, *p ⁇ 0.05 vs db/m; #p ⁇ 0.05vs db/db;
  • C is a liver microscopic examination of CD68 expression staining;
  • DG shows immunoblot analysis of protein expression of various inflammatory factors in the liver,
  • HJ shows the inflammatory factors of the liver Real-time quantitative PCR analysis of mRNA expression, *p ⁇ 0.05 vs db/m; #p ⁇ 0.05 vs db/db.
  • Figure 7 shows the effects of single intranasal administration of FGF1 WT and FGF1 ⁇ HS on long-term blood glucose levels, feeding and body weight in db/db mice, wherein (A) shows a single nasal administration to 42 days after administration. The blood glucose level, (B) shows the effect on food intake during this period, and (C) shows the change in body weight during this period.
  • Figure 8 shows the results of glucose tolerance test a single intranasal administration FGF1 WT after one month and FGF1 ⁇ HS db / db mice performed.
  • Figure 9 shows the results of microscopic examination of liver tissue of db/db mice 42 days after a single nasal administration of FGF1 ⁇ HS .
  • FGF1 ⁇ HS was cloned, expressed and purified by conventional means. Briefly, a cDNA fragment encoding full-length human wild-type FGF1 (abbreviated as FGF1 WT , 1-155) was cloned into the pET30a expression vector, followed by QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) The three mutations of Lys127Asp, Lys128Gln and Lys133Val were introduced. The expression vector carrying the mutant was transformed into Escherichia coli BL21 (DE3) and cultured at 37 ° C. When A 600 reached 0.5, incubation was continued for 4 hours by the addition of 1 mM IPTG.
  • FGF1 WT full-length human wild-type FGF1
  • SPR biosensor chip (BIAcore 2000system (GE Healthcare, Piscataway , NJ)
  • FGF1 WT FGF1 ⁇ HS heparin binding affinity
  • HPLC-MALS By HPLC-MALS analysis, HPLC (Waters 1500pump with 2498 UV detector and 2707 autosampler) coupled with MALS (Wyatt miniDawn-Treos and Optilab rEX) to compare FGF1 WT and FGF1 ⁇ HS to FGFR1c dimerization in the presence of heparin decasaccharide Induction ability. As a result, as shown in Fig.
  • heparin decasaccharide resulted in a FGF1 WT- FGFR1c dimerization complex whose molecular weight was 89.54 kDa by MALS (theoretical dimer value was 89.48 kDa); and FGF1 ⁇ HS- FGFR1c complex
  • the retention time is significantly extended and the molecular weight is only 57.64 kDa.
  • FGF1 WT and FGF1 ⁇ HS were added in the culture of 3T3L1 adipocytes and rat hepatoma cell line H4IIE. It was found that FGF1 ⁇ HS was weaker against FGFR A-loop tyrosine than FGF1 WT . The order of magnitude is closer to FFS19 and FGF21 (Figs. 1A and 1B). Despite this difference, FGF1 ⁇ HS still retains the effect of stimulating glucose uptake similar to FGF1 WT in 3T3L1 and H4IIE cells ( Figures 1C and 1D).
  • FGF1 WT and FGF1 ⁇ HS were compared with NIH 3T3 fibroblasts. The results are shown in Figure 1G. Compared with FGF1 WT , the mitogenic ability of FGF1 ⁇ HS was significantly lost; in addition, as shown in Figures 1H and 1I shown, FGF1 ⁇ HS phosphorylation ability of a-loop weak FGFR tyrosine at least 1 order of magnitude, and weakened ability to induce phosphorylation of FRS2 and ERK activation.
  • FGF1 WT and FGF1 ⁇ HS (0.5 mg/kg body weight) were administered to normal C57BL/6J mice daily for 3 months, and immunohistochemical staining and Western blotting were performed with PCNA and Ki67 (Fig. 2A), respectively (Fig. 2B).
  • the liver of the mouse was analyzed for hyperplasia.
  • FGF1 WT was observed to cause hyperplasia in mice, whereas FGF1 ⁇ HS was not proliferated in the control group relative to PBS.
  • mice C57BLKS/J-lepr db /lepr db ) and their control phenotypically normal mice (db/m) were purchased from the Model Animal Research Center of Nanjing University.
  • Fig. 4A Liver tissues from the long-term administered mice and their control mice were stained with different dyes, and the results are shown in Fig. 4A, in which unadministered db/db mice had significant hepatic steatosis, while FGF1 WT and Long-term administration of FGF1 ⁇ HS db/db mice alleviated the extent of hepatic steatosis.
  • Triglyceride levels in the liver of long-administered db/db mice were also significantly reduced, approaching phenotype-normal db/m mice (Fig. 4B).
  • a decrease in lipid accumulation in liver tissue indicates a decrease in lipogenesis and fat storage, and correspondingly, a large amount of lipogenic gene-expressing protein and its mRNA level in the liver tissue of long-term db/db mice are administered to db/m.
  • the closeness of the mouse (Fig. 4D-4J).
  • liver glycogen levels of the mice were stained by PAS.
  • db/db mice increased hepatic glycogen levels relative to db/m mice
  • long-term administration of DF with FGF1 WT and FGF1 ⁇ HS Hepatic glycogen levels were further increased in /db mice.
  • Quantitative testing also showed such results (Fig. 4C). It is shown that the hypoglycemic and insulin sensitivity of FGF1 WT and FGF1 ⁇ HS are achieved by increasing the synthesis and storage of hepatic glycogen.
  • db / db mice with the FGF1 WT and FGF1 ⁇ HS chronic administration db / db mice can be reduced levels of inflammatory cytokines IL-6 and TNF- ⁇ in plasma.
  • the liver of db/db mice had high levels of CD68 + macrophage infiltration (Fig. 6C), and both protein and RNA levels of CD68 and inflammatory cytokines IL-6 and TNF- ⁇ were up-regulated (Fig. 6D-6J).
  • the expression of macrophage infiltration, CD68 and inflammatory cytokines in the liver of db/db mice chronically administered with FGF1 WT and FGF1 ⁇ HS was significantly reduced (Fig. 6C-6J), almost reaching normal db /m mouse level.
  • FGF1 WT or FGF1 ⁇ HS 5 mg dissolve in 2 ml of medical injection grade gel aqueous solution, and then add 100 mg of poloxamer. After homogenization, freeze-drying to form a lyophilized powder. 5 mg of hydrocoagulylcholine and 50 mg of polyoxyethylene hydrogenated castor oil were dissolved in 2 ml of t-butanol, during which time the dissolution could be accelerated by ultrasonic or water bath heating. The above lyophilized powder is added to a t-butanol solution, uniformly dispersed, and then frozen in liquid nitrogen to form a solid, and then freeze-dried to obtain a dry powder, wherein the particle diameter is substantially between 100 nm and 1000 nm.
  • the dry powder can be administered by intranasal administration. Alternatively, the dry powder preparation can be resuspended in physiological saline immediately before use to obtain a nasal drop for the following experiment.
  • the nasal drops of FGF1 WT and FGF1 ⁇ HS were administered to the db/db mice nasally (the doses of FGF1 WT and FGF1 ⁇ HS were 300 ⁇ g/mouse, respectively), and the db/db and db/m mice of the control group were respectively.
  • the nasal drops prepared according to the above method but without FGF1 were administered, and then the blood glucose, body weight and feeding of each group of mice were measured every day (the results are shown in Fig. 7); after 1 month of administration, some mice were taken for sugar.

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Abstract

提供了FGF1突变体，其氨基酸序列在SEQ ID NO：1的基础上具有Lys127、Lys128和/或Lys133突变，其减少FGF1促细胞分裂和增殖副作用，但是具有降糖活性。还提供了该突变体的经鼻给药制剂。

Description

FGF1突变体和给药方法
本申请要求申请日为2016年8月31日的中国专利申请CN201610791561.7的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于蛋白质技术领域，具体而言，本发明涉及FGF1突变体，其减少FGF1促细胞分裂和增殖副作用，但是具有降糖等活性。
背景技术
成纤维细胞生长因子(Fibroblast Growth Factor，FGF)中共有18个家族成员，被分为5个旁分泌和1个内分泌亚家族。FGF作用的发挥则是通过与细胞表面具有酪氨酸激酶活性的FGF受体(FGF Receptor，FGFR)相互作用进而引起受体的二聚化并进一步激活下游级联信号来实现的。
其中，FGF1可以对db/db,ob/ob等2型糖尿病鼠模型产生重要的降糖和提高胰岛素敏感的功能，而且即使大剂量也不会造成低血糖的产生(可参见Nature Review Drug Discovery,2016；Nature Medicine,2016；Proc Natl Acad Sci U S A,2016；Diabetes,2016；Nature,2015等)。
然而，FGF1作为一个旁分泌蛋白，它通过和肝素以及FGFR相互作用形成紧密的三元复合物进而启动一系列下游复杂的信号通路，其中就包括促细胞分裂和增殖的效应，因此，频繁的注射FGF1无疑会在体内存在诱导增生甚至肿瘤产生的隐患。
本发明人为此经过长期研究，令人意外地发现了一种FGF1肝素结合位点突变体(简称为FGF1△HS)，其无需敲除整个肝素结合位点，而仅仅需要引入有限的突变，即可使得FGF1△HS仅能与FGFR形成较弱的二元复合物即可启动代谢调控的信号通路，而基本丧失了促细胞分裂和增殖的功能，同时，FGF1△HS又保持了降糖等药理作用。
发明内容
本发明要解决的技术问题在于提供新的FGF1突变体，其基本消除了野生型FGF1的促细胞分裂和增殖的能力，但是仍旧保留了FGF1的降血糖等功能，从而可以长期安全地给药。另外，本发明还提供了该突变体的编码基因、表达载体和宿主细胞以及医药用途等。
具体而言，在第一方面，本发明提供了FGF1突变体，其氨基酸序列在SEQ ID NO：1的基础上(仅)具有Lys127、Lys128和/或Lys133突变。本发明第一方面的突变体减少(优选消除)野生型FGF1(其氨基酸序列如SEQ ID NO：1所示)的促细胞分裂和增殖的能力，但是仍旧保留了降血糖等功能。
在本文中，突变的表示采用本领域技术人员所熟知的方式。例如，Lys127表示对第127位上的Lys进行突变；而Lys128Gln表示对第128位上的Lys进行突变，突变为Gln。突变可以是添加、缺失和/或取代。本领域技术人员知晓，通过改变已知多肽的编码基因序列并将其导入表达载体，可以制备出取代、添加和/或缺失了氨基酸残基的多肽或蛋白质，这些方法广泛记载于《分子克隆实验指南》等本领域公知的文献中。在本发明的具体实施方式中，突变是取代。
优选本发明第一方面的突变体(仅)具有Lys127Asp、Lys128Gln和/或Lys133Val突变。在本发明的具体实施方式中，该突变体(仅)具有Lys127Asp、Lys128Gln和Lys133Val突变。
在第二方面，本发明提供了编码本发明第一方面的突变体的多核苷酸。在本文中，多核苷酸可以是DNA形式，也可以是RNA形式，优选DNA形式。DNA形式包括天然cDNA和人工合成的cDNA，DNA可以是编码链或模板链。通过常规技术，如PCR方法、重组法或人工合成的方法，本领域技术人员可以很容易获得编码本发明的突变体的核酸分子多核苷酸或其片段。这些序列一旦获得，就可以将其克隆入载体，再转化或转染入相应的细胞，然后通过常规的宿主细胞进行增殖，从中分离得到大量的核酸分子。
在第三方面，本发明提供了一种载体，其含有本发明第二个方面所述的多核苷酸。在本文中，载体包括表达载体和克隆载体，是指本领域中常用的细菌质粒、粘粒、噬菌 粒、酵母质粒、植物细胞病毒、动物病毒及其它各种病毒载体。本发明中适用的载体包括但不限于：在细菌中表达用的载体(原核表达载体)、在酵母中表达用的载体(如毕赤酵母载体、汉逊酵母载体等)、在昆虫细胞中表达的杆状病毒载体、在哺乳动物细胞中表达用的载体(痘苗病毒载体、逆转录病毒载体、腺病毒载体、腺伴病毒载体等)、在植物中表达用的植物病毒载体以及在哺乳动物乳腺中表达用的各种载体。总之，只要能在宿主细胞中稳定复制，任何质粒和载体都可使用。优选表达载体包含选择标记基因，如细菌的氨苄青霉素抗性基因、四环素抗性基因、卡那霉素抗性基因、链霉素抗性基因、氯霉素抗性基因；酵母菌的新霉素抗性基因、Zeocin抗性基因，酵母菌的缺陷选择标志，如His,Leu,Trp等；真核细胞的新霉素抗性基因、Zeocin抗性基因、二氢叶酸还原酶基因及荧光蛋白标记基因等。在本发明的具体实施方式中，使用的是已经商品化的pET质粒。
在第四方面，本发明提供了一种细胞，如宿主细胞，其含有本发明第二个方面的多核苷酸。本发明的细胞可以含有本发明第三方面的载体，也可以用本发明第二方面的多核苷酸转化或转染。宿主细胞可以是原核细胞，也可以是真核细胞，如，细菌细胞、酵母细胞、植物细胞、昆虫细胞、哺乳动物细胞等。宿主细胞在转化或转染含本发明所述编码蛋白的基因序列后，即构成工程化细胞或细胞株，可用于生产所需融合蛋白。本领域技术人员能够恰当地选择适当的载体、宿主细胞，并熟知如何将载体高效地转化或转染入宿主细胞中，所用方法包括但不限于：氯化钙法、电穿孔法用于细菌细胞，电穿孔法和原生质体融合法用于酵母细胞，脂质体包裹、磷酸钙共沉淀、电融合法以及显微注射法用于哺乳动物细胞等真核细胞。优选本发明的宿主细胞是大肠杆菌BL21(DE3)。
在第五方面，本发明提供了药物组合物，其包含本发明第一方面的突变体以及药学上可接受的载体。本发明的药物组合物能治疗糖尿病、降血糖、治疗或预防肥胖症、抗炎或者用于降低炎性细胞因子水平，并且其减少(甚至消除)FGF1的促细胞增殖的副作用。因而，本发明的药物组合物能够长期安全地给药。在本文中，药学上可接受的载体指无毒的填充剂、稳定剂、稀释剂、佐剂或其他制剂辅料。根据本领域的公知技术，可以根据治疗目的、给药途径的需要将药物组合物制成各种剂型，包括干粉剂(如冻干粉 剂)、片剂、胶囊、滴剂、乳液剂、注射剂和喷剂等。该药物组合物可以是经脑给药，因此它可以是经脑给药制剂，如经脑注射剂。
在本发明的具体实施方式中，给药途径包括注射和经鼻给药，所以本发明第五方面的药物组合物是注射剂或经鼻给药制剂。其中，优选经鼻给药制剂包含脂质体包裹的本发明第一方面的突变体，它可以是喷鼻剂或滴鼻剂。
本发明的喷鼻剂可以是脂质体包裹的本发明第一方面的突变体(或野生型FGF1)的冻干粉剂。优选该冻干粉剂中脂质体包裹的本发明第一方面的突变体(或野生型FGF1)的粒径介于100nm-1000nm之间。在本发明的一个具体实施方式中，该冻干粉剂可以通过如下方法制备：
取本发明第一方面的突变体(或野生型FGF1)，溶解于高分子亲水胶材料的水溶液中，然后加入泊洛沙姆。混和均匀后，冷冻干燥，形成冻干粉；将脂质材料和聚氧乙烯氢化蓖麻油溶解在叔丁醇中；和，将上述冻干粉加入到叔丁醇溶液中，分散均匀后置于液氮冻结形成固体，然后冷冻干燥，即得干粉剂。优选其中，本发明第一方面的突变体(或野生型FGF1)、高分子亲水胶材料、泊洛沙姆、脂质材料和聚氧乙烯氢化蓖麻油的的重量比为0.5～50：0.1～10：10～1000：0.5～50：5～500，优选为1～5：0.5～2：50～200：1～5：20～100，最优选为5：1：100：5：50。
本发明的滴鼻剂可以是将上述冻干粉剂重悬于药学上可接受的滴鼻溶剂中而制备。
本发明第五方面的药物组合物优选是单位剂量形式的，如一粒、一滴、一针或一个胶囊，其包含单位剂量的本发明第一方面的突变体。在本文中，单位剂量指的是一次给药患者所需的药物量。优选本发明第五方面的药物组合物是经鼻给药制剂，其单位剂量为0.1～5mg，优选为0.3～1mg，如为0.5mg。该药物组合物的降糖效果持续至少一周、至少两周或至少一个月。本发明第五方面的药物组合物也可以是经脑给药制剂，其单位剂量为1～500μg，优选为3～100μg，如为10μg。
在第六方面，本发明提供了本发明第一方面的突变体在制备减少FGF1促细胞分裂和增殖副作用的药物中的应用。野生型FGF1具有促细胞分裂和增殖的作用，这容易导致增生，乃至癌变。而本发明的突变体及其药物能够显著降低(甚至基本消除)这样的 副作用，从而能够长期安全地给药。优选在本发明第六方面的应用中，药物是本发明第五方面所述的药物组合物。
本发明的突变体及其药物基本保留了FGF1的降糖的功能。因此，优选在本发明第六方面的应用中，药物用于降血糖。也优选在本发明第六方面的应用中，药物用于治疗糖尿病，尤其是2型糖尿病。
本发明的突变体及其药物基本保留了FGF1的降脂的功能。因此，优选在本发明第六方面的应用中，药物用于降血脂(如，甘油三酯)。也优选在本发明第六方面的应用中，药物用于治疗或预防肥胖症。
本发明的突变体及其药物还具有抗炎的作用，能够用于降低一些炎性细胞因子的水平。因此，优选在本发明第六方面的应用中，药物用于抗炎。也优选在本发明第六方面的应用中，药物用于降低炎性细胞因子(如，IL-6和/或TNF-α)水平。
在第七方面，本发明提供了对糖尿病患者持续降血糖的方法，其包括向该患者经鼻或经脑给药单位剂量的本发明第一方面的突变体一次。向糖尿病患者该患者经鼻给药单位剂量的野生型FGF1也能对该患者持续降血糖，
优选在本发明的第七方面中，经鼻给药可以用喷鼻剂或滴鼻剂给药，更优选经鼻给药的单位剂量为0.1～5mg，优选为0.3～1mg，如为0.5mg。另外优选在本发明的第七方面中，经脑给药向脑内注射给药，更优选经脑给药的单位剂量为1～500μg，优选为3～100μg，如为10μg。
本发明第一方面的突变体经鼻或经脑给药一次即可长期地续降血糖，优选持续是持续至少一周、至少两周或至少一个月。
优选在本发明第七方面的方法中，糖尿病是2型糖尿病。
本发明的有益效果在于：本发明的突变体基本丧失了促细胞分裂和增殖的功能，同时又保持了降糖等药理作用，所以比已经成药的FGF1更具安全用药的前景；基本保留FGF1的整体结构(并没有缺失整个HS结合区)，所以可以尽可能地利用现有FGF1的基因工程生产体系(包括载体、细胞和表达条件)，方便大规模生产；尤其是通过经鼻给药可以长期持续降血糖，因此其经鼻给药制剂更有成为上市药品的前景。
本发明引用了公开文献，这些文献是为了更清楚地描述本发明，它们的全文内容均纳入本文进行参考，就好像它们的全文已经在本文中重复叙述过一样。
为了便于理解，以下将通过具体的实施例对本发明进行详细地描述。需要特别指出的是，这些描述仅仅是示例性的描述，并不构成对本发明范围的限制。依据本说明书的论述，本发明的许多变化、改变对所属领域技术人员来说都是显而易见了。
附图说明
图1显示了FGF1WT和FGF1△HS各种体外刺激的实验结果，其中，(A)为FGF1WT、FGF1△HS和野生型FGF21(FGF21WT)在3T3-L1脂肪细胞中对FGFR,FGFR底物2(FRS2)和MAPK途径(ERK1/2)的活化的免疫印迹图；(B)为FGF1WT、FGF1△HS和野生型FGF19(FGF19WT)在大鼠肝细胞瘤细胞系H4IIE中对FGFR,FGFR底物2(FRS2)和MAPK途径(ERK1/2)的活化的免疫印迹图；(C)为FGF1WT、FGF1△HS和FGF21WT刺激3T3-L1脂肪细胞所产生的细胞糖摄入情况；(D)为FGF1WT、FGF1△HS和FGF19WT刺激大鼠肝细胞瘤细胞系H4IIE产生的细胞糖摄入情况；(E)为SPR检测图谱；(F)为HPLC-MALS检测图谱(G)显示了FGF1WT和FGF1△HS为对NIH 3T3细胞的增殖效应，*p<0.05vs PBS缓冲液(对照)；(H)为FGF1WT和FGF1△HS在NIH 3T3细胞中对FGFR、FRS2和MAPK途径(ERK1/2)的活化的免疫印迹图；(I)为100ng/ml FGF1WT和FGF1△HS在NIH 3T3细胞中对FGFR、FRS2和MAPK途径(ERK1/2)的活化的免疫印迹图。
图2显示了FGF1WT和FGF1△HS对体内肝组织的促有丝分裂活性，其中，(A)为用增殖细胞核抗原(PCNA)和Ki67染色的鼠肝组织切片镜检图；(B)为用FGF1WT和FGF1△HS长期给药的小鼠的肝组织蛋白表达的免疫印迹分析，*p<0.05vs对照(用PBS给药)，#p<0.05vs FGF1WT。
图3显示了FGF1WT和FGF1△HS对db/db小鼠中血糖和胰岛素敏感性的影响，其中，(A)为单次注射FGF1WT和FGF1△HS对db/db小鼠中24小时血糖水平的影响，*p<0.05vs db/m；#p<0.05vs db/db.(B)显示了FGF1WT和FGF1△HS对db/db小鼠中血糖降低的剂量 依赖性影响；(C)为单次注射FGF1WT和FGF1△HS后6小时进行的GTT的血糖水平，(D)为单次注射FGF1WT和FGF1△HS后6小时进行的GTT的AUC，*p<0.05vs db/m；#p<0.05vs db/db；(E)为单次注射FGF1WT和FGF1△HS后6小时进行的ITT的血糖水平，(F)为单次注射FGF1WT和FGF1△HS后6小时进行的ITT的AUC，*p<0.05vs db/m；#p<0.05vs db/db；(G)为用FGF1WT和FGF1△HS经28天给药的db/db小鼠的血糖水平，*p<0.05vs db/m；#p<0.05vs db/db。
图4显示了FGF1WT和FGF1△HS对db/db小鼠中肝脏脂和糖代谢的长期影响，其中，(A)为用H&E、Oil Red O和PAS染色的鼠肝组织切片镜检图；(B)为用FGF1WT和FGF1△HS经28天给药的db/db小鼠的肝组织中的甘油三酯含量，(C)为用FGF1WT和FGF1△ HS经28天给药的db/db小鼠的肝组织中的糖原含量，*p<0.05vs db/m；#p<0.05vs db/db；(D-G)显示了肝脏各蛋白质表达的免疫印迹分析，(H-J)显示了肝脏各mRNA表达的实时定量PCR分析，*p<0.05vs db/m；#p<0.05vs db/db。
图5显示了FGF1WT和FGF1△HS对db/db小鼠中脂肪组织重塑和脂代谢的长期影响，其中，(A)为用H&E染色的鼠白色脂肪组织(WAT)切片镜检图；(B)为用H&E染色的鼠棕色脂肪组织(BAT)切片镜检图；(C-G)显示了WAT各mRNA表达的实时定量PCR分析，*p<0.05vs db/m；#p<0.05vs db/db。
图6显示了FGF1WT和FGF1△HS对db/db小鼠全身和肝脏炎症的长期影响，其中，(A)显示了用FGF1WT和FGF1△HS经28天给药的db/db小鼠血浆中的IL-6浓度，(B)显示了用FGF1WT和FGF1△HS经28天给药的db/db小鼠血浆中的TNF-α浓度，*p<0.05vs db/m；#p<0.05vs db/db；(C)为对CD68表达染色的肝组织镜检图；(D-G)显示了肝脏各炎性因子的蛋白质表达的免疫印迹分析，(H-J)显示了肝脏各炎性因子的mRNA表达的实时定量PCR分析，*p<0.05vs db/m；#p<0.05vs db/db。
图7显示了单次鼻腔给药FGF1WT和FGF1△HS对db/db小鼠的长期血糖水平、摄食和体重的影响，其中，(A)显示了单次鼻腔给药至给药后42天的血糖水平，(B)显示了在此期间对摄食量的影响，(C)显示了在此期间体重的变化情况。
图8显示了单次鼻腔给药FGF1WT和FGF1△HS后一个月对db/db小鼠进行的糖耐测 试的结果。
图9显示了单次鼻腔给药FGF1△HS后42天db/db小鼠肝脏组织的镜检结果。
具体实施方式
以下通过实施例进一步说明本发明的内容。如未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段和市售的常用仪器、试剂，可参见《分子克隆实验指南(第3版)》(科学出版社)和CFDA的相关试验指引以及相应仪器和试剂的厂商说明书等参考。
实施例1本发明的FGF1△HS及其体外活性研究
通过常规手段克隆、表达并纯化FGF1△HS。简而言之，将编码全长人野生型FGF1(简称为FGF1WT,第1-155位)的cDNA片段克隆入pET30a表达载体中，然后用QuikChange XL定点突变试剂盒(Stratagene,La Jolla,CA)导入Lys127Asp、Lys128Gln和Lys133Val这三个突变。将带有突变体的表达载体转化入Escherichia coli BL21(DE3)中，于37℃培养，当A600达到0.5时，加入1mM IPTG继续培养4小时。菌体裂解后用依次阳离子交换层析柱和凝胶排阻层析柱(GE Healthcare,Piscataway,NJ)纯化，获得纯度>98％的FGF1△HS。其他对照蛋白也可以用相似方法制备。
通过SPR生物感应芯片(BIAcore 2000system(GE Healthcare,Piscataway,NJ))检测，相比于FGF1WT，FGF1△HS的肝素结合亲和性得到了显著的丧失(参见图1E)。
通过HPLC-MALS分析，其中HPLC(Waters 1500pump with 2498UV detector and2707autosampler)偶联MALS(Wyatt miniDawn-Treos and Optilab rEX)，来比较在肝素十糖的存在下，FGF1WT和FGF1△HS分别对FGFR1c二聚化的诱导能力。结果如图1F所示，肝素十糖导致了FGF1WT-FGFR1c二聚化复合物，其分子量通过MALS测得为89.54kDa(理论二聚体值为89.48kDa)；而FGF1△HS-FGFR1c复合物的保留时间明显延长，分子量仅为57.64kDa。
在培养3T3L1脂肪细胞和大鼠肝细胞瘤细胞H4IIE时加入不同浓度的FGF1WT和FGF1△HS，发现相对于FGF1WT，FGF1△HS对FGFR的A-loop酪氨酸的磷酸化能力弱了两 个数量级，而对FRS2α-RAS-MAPK途径的活化更接近于FGF19和FGF21(图1A和1B)。尽管如此不同，但是在3T3L1和H4IIE细胞中，FGF1△HS仍旧保留了类似于FGF1WT的刺激葡萄糖摄取的效应(图1C和1D)
用NIH 3T3成纤维细胞比较FGF1WT和FGF1△HS的促有丝分裂能力，结果如图1G所示，相比于FGF1WT，FGF1△HS的促有丝分裂能力得到了显著的丧失；另外如图1H和1I所示，FGF1△HS对FGFR的A-loop酪氨酸的磷酸化能力弱了至少1个数量级，并且减弱了诱导FRS2磷酸化和ERK活化的能力。
实施例2本发明的FGF1△HS的体内活性研究
(1)正常小鼠
每天对正常的C57BL/6J小鼠给药FGF1WT和FGF1△HS(0.5mg/kg体重)，持续3个月，分别用PCNA和Ki67(图2A)免疫组化染色和Western blotting(图2B)对小鼠的肝脏分析增生情况。FGF1WT被观察到能导致小鼠增生，而FGF1△HS相对于PBS的对照组，没有出现增生。
(2)FGF1WT和FGF1△HS对db/db小鼠的血糖水平和胰岛素敏感性的影响
糖尿病模型(db/db)小鼠(C57BLKS/J-leprdb/leprdb)和其对照的表型正常的小鼠(db/m)均购自南京大学模型动物研究中心。
在db/db小鼠中，快速注射FGF1WT和FGF1△HS(0.5mg/kg体重)都能显著降低血糖水平，该效果都基本能持续到注射后的24小时(图3A)，而且，该效果都是剂量依赖性的(图3B)。
在糖耐测试(GTT)中，快速注射FGF1WT和FGF1△HS的db/db小鼠都能保持较低的血糖水平(图3C和3D)；而在胰岛素耐受测试(ITT)中,注射FGF1WT和FGF1△HS的db/db小鼠都显示出了显著的胰岛素敏感性的改善(图3E和3F)。
通过隔天注射db/db小鼠0.5mg/kg的FGF1WT和FGF1△HS，持续4周，来观察长期给药的效果。结果如图3G所示，FGF1WT和FGF1△HS都能使db/db小鼠的血糖水平在4周恢复正常。
(3)FGF1WT和FGF1△HS对db/db小鼠的肝脏脂和糖代谢的长期影响
前述长期给药的小鼠及其对照小鼠中取肝脏组织用不同染料染色，结果如图4A所示，其中未给药的db/db小鼠有着显著的肝脂肪变性，而用FGF1WT和FGF1△HS长期给药的db/db小鼠都减轻了肝脂肪变性的程度。
长期给药的db/db小鼠的肝脏的甘油三酯水平也显著降低，接近了表型正常的db/m小鼠(图4B)。肝组织中的脂积累的减少表明脂肪生成和脂肪储存的减少，相应地，在长期给药的db/db小鼠的肝组织中大量脂肪生成基因的表达蛋白及其mRNA水平均向db/m小鼠的靠拢(图4D-4J)。
小鼠的肝糖原水平通过PAS染色，结果如图4A所示，db/db小鼠相对于db/m小鼠提高了肝糖原水平，而用FGF1WT和FGF1△HS长期给药的db/db小鼠的肝糖原水平进一步增加了。定量检测也显示了这样的结果(图4C)。表明FGF1WT和FGF1△HS的降糖和提高胰岛素敏感性是通过增加肝糖原的合成和储存来实现的。
(4)FGF1WT和FGF1△HS对db/db小鼠的脂肪重塑和脂代谢的长期影响
肥胖症和2型糖尿病均油增多的脂肪储存和白色脂肪细胞(WAT)扩充的特征，而用FGF1WT和FGF1△HS长期给药的db/db小鼠都减少了白色和棕色脂肪细胞(BAT)的大小(图6A和6B)；相应的WAT中的大量脂肪生成基因的表达也向正常水平靠拢(图6C-6G)。
(5)FGF1WT和FGF1△HS对db/db小鼠的全身性炎症的长期影响
如图6A和6B所示，用FGF1WT和FGF1△HS长期给药的db/db小鼠都能降低血浆中炎性细胞因子IL-6和TNF-α的水平。db/db小鼠的肝脏中具有高的CD68+巨噬细胞浸润水平(图6C)，并且CD68和炎性细胞因子IL-6和TNF-α的蛋白和RNA水平都被上调(图6D-6J)；而用FGF1WT和FGF1△HS长期给药的db/db小鼠的肝脏中的巨噬细胞浸润、CD68和炎性细胞因子的表达都显著降低(图6C-6J)，几乎达到正常db/m小鼠的水平。
实施例3本发明的给药制剂
取FGF1WT或FGF1△HS 5mg，溶解于2ml医用注射级凝胶水溶液中，然后加入100mg 泊洛沙姆。混和均匀后，冷冻干燥，形成冻干粉。将5mg氢化凝脂酰胆碱和50mg聚氧乙烯氢化蓖麻油溶解在2ml叔丁醇中，期间可以采用超声或水浴加热的处理方法来加速溶解。将上述冻干粉加入到叔丁醇溶液中，分散均匀后置于液氮冻结形成固体，然后冷冻干燥，即得干粉剂，其中粒径基本介于100nm-1000nm之间。该干粉剂可以喷入鼻腔给药。另外，可将该干粉制剂在临用前重悬于生理盐水中，得到滴鼻剂，用于以下实验。
分别取FGF1WT和FGF1ΔHS的滴鼻剂向db/db小鼠鼻腔给药一次(FGF1WT和FGF1ΔHS的剂量分别为300μg/只小鼠)，对照组的db/db和db/m鼠分别给药按以上方法制备但不含FGF1的滴鼻剂，然后每天检测各组小鼠的血糖、体重和摄食(结果如图7所示)；给药I个月后，取部分小鼠进行糖耐测试)即，于-30min测初始血糖，0min后腹腔注射葡萄糖(18mmol/kg)，并在0min、15min、30min、60min和120min分别尾部取血，测定葡萄糖浓度)(结果如图8所示)；最后于第43天处死，观察各组肝脏组织的改善情况(结果如图9所示)。
如图7所示，向db/db小鼠鼻腔给药一次FGF1WT和FGF1△HS都能显著降低血糖水平，而且该效果能持续到给药后的42天甚至更长时间，而此期间对小鼠的摄食量和体重基本没有影响。
如图8所示的糖耐测试结果表明，相比对照组，单次鼻腔给药后一个月，给药了FGF1WT和FGF1△HS的db/db小鼠仍旧能保持较低的血糖水平，具有很好的葡萄糖耐受性。
如图9所示，相比对照组，单次鼻腔给药FGF1△HS42天后，肝脏脂滴变小，脂肪化程度明显改善。
结果表明，FGF1WT和FGF1△HS的经鼻腔给药的制剂具有卓越的降糖效果，而且作用效果能持续超过一个多月，对摄食和体重影响不大，尤其是FGF1△HS还没有引发增生的效应，更具有很好的安全性，因此更具有提供高效的2型糖尿病防治方法的前景。

Claims (20)

  1. FGF1突变体，其氨基酸序列在SEQ ID NO：1的基础上具有Lys127、Lys128和/或Lys133突变。
  2. 权利要求1所述的突变体，其具有Lys127Asp、Lys128Gln和/或Lys133Val突变。
  3. 权利要求2所述的突变体，其氨基酸序列在SEQ ID NO：1的基础上具有Lys127Asp、Lys128Gln和Lys133Val突变。
  4. 编码权利要求1～3之一所述的突变体的多核苷酸。
  5. 载体或细胞，其包含权利要求4所述的多核苷酸。
  6. 权利要求1～3之一所述的突变体在制备减少FGF1促细胞分裂和增殖副作用的药物中的应用。
  7. 权利要求6所述的应用，其中药物用于抗炎或者用于降低炎性细胞因子(如，IL-6和/或TNF-α)水平。
  8. 权利要求6所述的应用，其中药物用于治疗糖尿病，尤其是2型糖尿病。
  9. 权利要求6所述的应用，其中药物用于降血糖。
  10. 权利要求6所述的应用，其中药物用于治疗或预防肥胖症。
  11. 药物组合物，其包括权利要求1～3之一所述的突变体以及药学上可接受的载体。
  12. 权利要求11所述的组合物，其是经鼻给药制剂。
  13. 权利要求12所述的组合物，其中经鼻给药制剂包含脂质体包裹的权利要求1～3之一所述的突变体。
  14. 权利要求12所述的组合物，其中经鼻给药制剂是喷鼻剂或滴鼻剂。
  15. 权利要求12所述的组合物，其降糖效果持续至少一周、至少两周或至少一个月。
  16. 权利要求12所述的组合物，其单位剂量为0.1～5mg，优选为0.3～1mg，如为0.5mg。
  17. 权利要求6～10之一所述的应用，其中药物是权利要求11至16之一所述的药物组合物。
  18. 对糖尿病患者持续降血糖的方法，其包括向该患者经鼻给药0.1～5mg(优选为 0.3～1mg，如为0.5mg)的权利要求1或2所述的突变体一次。
  19. 权利要求18所述的方法，其中持续是持续至少一周、至少两周或至少一个月。
  20. 权利要求18所述的方法，其中糖尿病是2型糖尿病。
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